Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.12/May-2019/6.pdf Open Access

Isolation and molecular characterization of spp. in sheep and goats in Egypt

Mounier M. Abdel Halium1, Fayez A. Salib1, S. A. Marouf2 and Emil S. Abdel Massieh1

1. Department of Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt; 2. Department of Microbiology and Mycology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Corresponding author: Emil S. Abdel Massieh, e-mail: [email protected] Co-authors: MMAH: [email protected], FAS: [email protected], SAM: [email protected] Received: 04-12-2018, Accepted: 15-03-2019, Published online: 13-05-2019 doi: 10.14202/vetworld.2019.664-670 How to cite this article: Abdel Halium MM, Salib FA, Marouf SA, Abdel Massieh ES (2019) Isolation and molecular characterization of Mycoplasma spp. in sheep and goats in Egypt, Veterinary World, 12(5): 664-670.

Abstract Background and Aim: Different species of Mycoplasma are associated with many pathological problems in small including respiratory manifestation, this problem results in significant losses, especially in African countries. This study aimed to (I) study some epidemiological aspects of Mycoplasma species infections in Egyptian sheep and goats at Giza Governorate, (II) diagnosis of Mycoplasma species affections using bacterial isolation and identification, (III) apply the polymerase chain reaction (PCR) for typing of different Mycoplasma species, and (IV) illustrate the phylogenetic tree for the isolated Mycoplasma species and other species from GenBank using the purified PCR product. Materials and Methods: A total of 335 samples were collected from sheep and goats from Giza Governorate in Egypt as 142 nasal swabs from clinically affected animals, 167 pneumonic , 18 samples from tracheal bifurcation, and 8 samples by bronchial wash were cultured on pleuropneumonia-like organisms (PPLOs) media for cultivation of Mycoplasma species. PCR and sequencing and phylogenetic analysis were adopted to identify and classify the isolated Mycoplasma species. Results: A total of 24 Mycoplasma isolates were isolated on PPLO media, identified by biochemical tests, and confirmed and typed by PCR using specific primers. 10 isolates were confirmed as Mycoplasma arginini, four isolates as Mycoplasma ovipneumoniae by PCR, and 10 isolates as undifferentiated Mycoplasma species. A purified isolate of M. arginini and M. ovipneumoniae was sequenced and phylogenetic analysis was illustrated. Conclusion: M. arginini and M. ovipneumoniae are prevalent in Egyptian sheep and goats. Further studies on M. arginini are required due to its high frequency of isolation from pneumonic sheep and goats and also from animals suffer from different respiratory manifestations. Keywords: goats, Mycoplasma, polymerase chain reaction, sequencing and phylogenetic analysis, sheep. Introduction and mastitis [5]. Mycoplasma species commonly Mycoplasma belongs to a group of associated with pneumonia in small ruminants are named which characterized by its min- Mycoplasma ovipneumoniae, Mycoplasma arginini, ute genome size and perpetually devoid of the cell Mycoplasma capri, Mycoplasma capripneumoniae, wall [1]. Different Mycoplasma species are accompa- and Mycoplasma capricolum [6]. M. arginini is fre- nying by many diseases and problems in both mam- quently isolated with M. ovipneumoniae from cases of malian and avian species [2], sheep and goats (poor atypical pneumonia in sheep and goats [5] and other man’s cow) are economically critical in many coun- cases with consolidation [7]. M. arginini is also tries including Egypt as consumed for meat, wool, and isolated from other locations such as genital organs, milk production, more than 10% of meat production eyes, and ears [8]; in addition, isolation of M. arginini in Egypt is from sheep and goats [3]. from pathological cases in human [9,10] gives suspi- Mycoplasma infections leading to a great eco- cion that M. arginini may have zoonotic importance. nomic loss due to it cause high morbidity and mor- In Egypt, different Mycoplasma species have been tality rates in sheep and goats populations in African isolated including M. arginini, M. ovipneumoniae, countries including Egypt, European countries, and and Mycoplasma agalactiae [11]. India [4]. Mycoplasma causes various clinical man- The respiratory problems are considered as mul- ifestations as pneumonia, conjunctivitis, arthritis, tifactorial disease where interaction between different microbial agents such as bacteria (Pasteurella and Copyright: Abdel Halium, et al. Open Access. This article is Mycoplasma), viruses (PI3, reovirus, and adenovi- distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ rus), and fungi and also other factors as host defense by/4.0/), which permits unrestricted use, distribution, and mechanism and environmental factors including cli- reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the matic conditions and stress of transportations lead to Creative Commons license, and indicate if changes were made. increase the incidence of those problems [12]. The Creative Commons Public Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data Mycoplasma is highly fastidious microorganism made available in this article, unless otherwise stated. required very precise media to develop in vitro, the

Veterinary World, EISSN: 2231-0916 664 Available at www.veterinaryworld.org/Vol.12/May-2019/6.pdf low ability of Mycoplasma to form macromolecules Postmortem examination needed for their growth refers to their evolutionary Lung tissues, tracheas, and tracheal bifurcation development from other bacteria; it is highly sus- of slaughtered sheep and goats were examined to pected that many exist in nature, how- detect and record different pathological changes [15]. ever, have not been isolated due to their hard growth Epidemiological investigation in vitro on artificial media [1]. The prevalence of isolation of different Lately, the high development of polymerase Mycoplasma species in the examined sheep and goats chain reaction (PCR) technique makes the detection was recorded according to species, ages, sexes, and of different species of Mycoplasma using specific season of the year [16]. primers much simpler, till now, PCR remains the most valuable and rapid method for detecting specific spe- Bacterial isolation and identification of Mycoplasma species cies of Mycoplasma [13]. Moreover, the amplified For nasal swab from living sheep and goats with purified PCR products of many genes from differ- respiratory manifestation, sterile cotton swabs were ent strains can be sequenced, offering the chance for used to collect nasal swabs from nostrils of affected whole or partial typing of various species and strains at a more accurate level allowing molecular epidemi- sheep and goats, and they were taken on pleuro- ological studies and research. pneumonia-like organisms (PPLOs) broth placed in The molecular epidemiological analysis allows an icebox and submitted directly to the laboratory. the genotyping of different strains and has used in the Serial dilutions from nasal swab were formed and tracing, control, and prevention of some diseases; also, incubated on PPLO broth of pH 7.4-7.6 with 15% molecular typing of some strains such as M. arginini heat-inactivated swine serum, 10% fresh yeast extract, would help in strain differentiation and then know the and 0.0005 g/ml thallium acetate at 37°C for 3-7 days, suspected pathological importance of these strains [8]. then cultured on solid PPLO media by running drop This study aimed to (I) study some epidemiological technique [17]. aspects of Mycoplasma species infections in Egyptian For bronchial washes, approximately 1 cm long sheep and goats at Giza Governorate, (II) diagnosis of skin incision was done with a surgical scalpel over the Mycoplasma species affections using bacterial isola- midpoint of the trachea under local anesthesia of 2% tion and identification, (III) apply the PCR for typing lidocaine with epinephrine, a 16-gauge hypodermic of different Mycoplasma species, and (IV) illustrate needle was inserted into the tracheal lumen. A catheter the phylogenetic tree for the isolated Mycoplasma equipped with two-way lumen connect valve was then species and other species from GenBank using the inserted through the needle up to bronchial bifurca- purified PCR product. tion; approximately 20-50 ml of Hank’s balanced salt solution was injected through the catheter and then Materials and Methods immediately aspirated with syringe. The debris was Ethical approval then removed by low-speed centrifugation; and then, Animal ethical approval was obtained from the samples were cultured on PPLO broth for 3-7 days Institutional Animal Care and Use Committee, Cairo at 37°C and then cultured on solid PPLO by running University (II F 23 18). drop technique [18,19]. Animals and samples For lung tissues and tracheal bifurcations from A total of 246 sheep and 89 goats of different ages, slaughtered sheep and goats, 167 samples from pneu- sexes, and breeds suffering from respiratory manifes- monic areas and 18 samples from tracheal bifurcation tations were examined clinically and bacteriology for were aseptically taken and placed in sterile plates kept isolation and identification of Mycoplasma species. in an icebox and were submitted to the laboratory. The The present investigation was carried out outer surface of the pneumonic lungs was first seared between December 2015 and November 2016. with a heated spatula before cutting the inner surface A total of 142 nasal swabs (58 sheep and 84 of the lungs. Samples were taken either from bron- goats) and eight bronchial washes (seven sheep and chioles of each lobe of cut lung sections with micro one goat) were collected from sheep and goats suffer- tipped swabs or from lung homogenates by blending ing from respiratory manifestations in different local- lung tissue with Hank’s balanced salt solution fol- ities at Giza Governorate. A total of 167 lung tissues lowed by removal of debris by low-speed centrifu- (165 sheep and two goats) and 18 tracheal bifurcations gation., Samples were then cultured on PPLO broth (16 sheep and two goats) were collected from slaugh- for 3-7 days at 37°C and then cultured on solid PPLO tered sheep and goats at Monieb abattoir. by running drop technique; filter syringe of 0.45 nm Clinical examination pore diameter was used for filtration of cultured broth Clinical signs of different respiratory manifes- media before culturing on solid media. tations in examined sheep and goats were recorded, Mild turbidity in liquid media of cultured sam- a general examination of affected animals includ- ples and fried egg appearance after examination of ing body temperature was measured, cough test was plates under the microscope indicates mullicates applied, and chest auscultation was also adopted [14]. growth [20]. 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PCR mucopurulent, and purulent), cough, and abnormal For detection and typing of Mycoplasma spe- chest sound with auscultation (Figure-1a and b). cies using PCR, positive cultures in liquid broth were Postmortem examination centrifuged at 15,000 rpm for 10 min, and the pellet The examined lungs, tracheas, and tracheal bifur- was resuspended in 300 µl of sterile distilled water, cation showed different pathological lesions including 300 µl TNES buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 10 mM Tris-ethylenediamine tetra-acetic acid, congestion and edema with red and gray hepatization, and 0.2% sodium dodecyl sulfate) and proteinase K hemorrhages in the trachea and tracheal bifurcation, (200 µg/ml) were then added to the bacterial suspen- and different degrees of pleurisy (Figure-1c). sion and kept at 56°C for 1 h. The suspension was Some epidemiological data heated at 95°C for 10 min to inactivate proteinase K The results of isolation of different Mycoplasma using a heat block method [20], the primer sequences, species in the examined sheep and goats in relation to conditions of PCR, and the amplicon size with refer- sex, season, and age are illustrated in Tables-2 and 3. ences are illustrated in Table-1 [2,5,10,20]. Bacterial isolation and identification of Mycoplasma DNA sequencing and phylogenetic analysis species For PCR product purification and sequencing, The collected samples were considered positive QIAquick PCR Product Extraction Kit (Qiagen Inc., to Mycoplasma species when bacterial growth was Valencia, CA) was used for purification of the PCR observed, and it was indicated by turbidity in PPLO product directly while a purified PCR product was broth and fried egg appearance colonies in the agar sequenced in the forward and/or reverse directions on an Applied Biosystems 3130 automated DNA Sequencer (ABI, 3130, USA), using already avail- able BigDye Terminator V3.1 sequencing kit (Perkin- Elmer/Applied Biosystems, Foster City, CA) [21]. A BLAST® analysis (Basic Local Alignment Search Tool) was initially performed to establish a sequence identity to GenBank accessions. A com- b parative analysis of sequences was performed using the CLUSTAL W multiple sequence alignment pro- gram, version 1.83 of Meg Align module of Lasergene DNAStar software pairwise, which was designed by Thompson et al. [22] and phylogenetic analyses were done using maximum likelihood, neighbor-joining, and maximum parsimony in MEGA6 [23]. c d Results Figure-1: (a) Buck suffers from different respiratory Clinical examination manifestations with nasal and ocular discharges. (b) Goats Living animals were examined clinically; ani- suffer from severe respiratory distress with nasal and mals were suffered from different signs of respira- ocular discharge (corneal opacity). (c) Pneumonic sheep lung with areas of red hepatization, especially at the cranial tory manifestations including fever 40-42°C with lobe. (d) Colonies of Mycoplasma appear as a fried egg depression, nasal and ocular discharges (mucoid, under the microscope.

Table-1: PCR primers, conditions, and amplicon size.

Species Primers Annealing Amplicon References temperature size Mycoplasma genus GPO3F: 5’‑TGGGGAGCAAACAGGATTAGATACC‑3’ At 53°C for 15 s 278 [20] MGSO: 5’‑ TGCACCATCTGTCACTCTGTTAACCTC‑3 M. arginini MAGF: 5’‑GCA TGG AAT CGCATG ATT CCT‑3’ 46°C for 60 s 525 [10] GP4R: 5’‑GGT GTT CTT CCTTAT ATC TAC GC‑3’ M. ovipneumoniae MOVPF: 5’‑GTT GGT GGC AAA AGTCACTAG‑3’ At 62°C for 90 s 418 [2] MOVPR: 5’‑CTT GACATC ACT GTT TCG CTG‑3’ Mycoplasma capricolum spe‑F: 5’‑ATCATTTTTAATCCCTTCAAG‑3’ 47°C for 15 s 316 [20] subspecies capripneumoniae spe‑R: 5’‑TACTATGAGTAATTATAATATATGCAA‑3’ Mycoplasma capricolum MCCPL1‑L: 5’‑AGACCCAAATAAGCCATCCA‑3’ At 47°C for 15 s 1350 [5] subspecies capricolum MCCPL1‑R: 5’‑CTTTCACCGCTTGTTGAATG‑3’ P4: 5’‑ACTGAGCAATTCCTCTT‑3’ At 46°C for 90 s 195 [5] subspecies Capri p5: 5’‑TTAATAAGTCTCTATATGAAT‑3’ M. agalactiae Mag‑F: 5’‑CCTTTTAGATTGGGATAGCGGATG‑3’ 60°C for 60 s 360 [5] Mag‑R: 5’‑CCGTCAAGGTAGCGTCATTTCCTAC‑3’ PCR=Polymerase chain reaction, M. arginine=Mycoplasma arginine, M. agalactiae=Mycoplasma agalactiae, Mycoplasma ovipneumoniae=M. ovipneumoniae

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Table-2: Number and percentage of positive samples with Mycoplasma species in examined sheep and goats in relation to sex and season.

Samples Sex Sheep Goats Season Season Total Winter Spring Summer Autumn Winter Spring Summer Autumn Nasal Male 12 1 10 1 8 1 9 0 17 2 14 1 9 1 14 1 93 8 swabs Female 6 0 4 0 3 0 6 1 9 1 8 1 6 0 7 0 49 3 Bronchial Male 2 1 1 0 1 0 1 0 1 0 ‑ ‑ ‑ ‑ ‑ ‑ 6 1 wash Female 1 0 ‑ ‑ ‑ ‑ 1 0 ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ 2 0 Tracheal Male 4 1 3 0 2 0 3 0 1 1 ‑ ‑ ‑ ‑ ‑ ‑ 13 2 bifurcation Female 1 0 1 0 ‑ ‑ 2 1 ‑ ‑ ‑ ‑ ‑ ‑ 1 0 5 1 Lung Male 31 2 28 2 21 1 23 1 1 0 ‑ ‑ ‑ ‑ ‑ ‑ 104 6 Female 21 1 16 1 13 0 12 0 ‑ ‑ ‑ ‑ ‑ ‑ 1 1 63 3 Total 78 6 63 4 48 2 57 3 29 4 22 2 15 1 23 2 ♂ 216 17 7.78% ♂ 119 7 5.88% Percentage of 7.7 6.35 4.16 5.26 13.8 9.1 6.67 8.7 positive samples

Table-3: Distribution of Mycoplasma species in examined sheep and goats in relation to age.

Age Sheep Goats Total Number of Number of Number of Number of Number of Percentage of samples positive samples samples positive samples positive samples positive samples <15 months 102 7 29 4 11 8.37 From 15 months to 79 5 33 3 8 7.14 21 months From 21 months to 34 2 14 1 3 6.25 27 months >27 months 31 1 13 1 2 4.54 plates (Figure-1d). Bacterial growth was observed M. capricolum subspecies capricolum, Mycoplasma in 24 samples (15 from sheep and nine from goats). mycoides subspecies capri, and M. agalactiae) Biochemically, all the 24 samples showed zones of using species-specific primers and were nega- growth inhibition around digitonin discs 5 mm in tive, the 10 samples considered as undifferentiated diameter or more, 14 were glucose fermentation test Mycoplasma species (Table-4). positive, and 10 samples were arginine hydrolysis The high-frequency rate of isolation of M. argi- positive. nini from sheep and goats suffering from respiratory PCR manifestations and pneumonic lungs makes us more The total of 24 culture-positive isolates was interested, amplified purified PCR product of one confirmed as belonging to the Mycoplasma genus isolate was subjected to sequencing analysis and the (Figure-2a) by the group-specific PCR, which pro- sequence then was submitted to NCBI GenBank tak- duced specific bands with the molecular size of ing accession number of MH685445 and also, purified 278 bp. PCR product of one isolate of M. ovipneumoniae was In M. arginini-specific PCR amplification of the subjected to sequencing analysis and the sequence samples, six of the sheep isolates and four of the goat iso- then submitted to NCBI GenBank taking accession lates produced positive products with the approximate number of MK045665. molecular size of 525 bp (Figure-2b). The isolation per- Phylogenetic analysis centages of M. arginini were, therefore, calculated as A phylogenetic tree (Figure-3a and b) was 40% (6/15) in sheep and 44.4 (4/9) in goats. formed based on 16S gene sequence of one purified In M. ovipneumoniae-specific PCR amplifica- strain of each species from the Egyptian isolates and tion of the samples, three of the sheep isolates and one other isolates of M. arginini and M. ovipneumoniae of the goat isolates produced positive products with isolated from different countries and different species the approximate molecular size of 418 bp (Figure-2c). (from GenBank). Sequencing of 16S genes of M. argi- The isolation percentages of M. ovipneumoniae in nini and M. ovipneumoniae Egyptian isolate and other related to total Mycoplasma isolates were, therefore, isolates from other countries showed no significant calculated as 20% (3/15) in sheep and 11.11 (1/9) in difference between them. goats. The other 10 isolates which were positive with Discussion the general primer of Mycoplasma genus were tested Mycoplasmas of different species have been against (M. capricolum subspecies capripneumoniae, implicated in causing various diseases in sheep and

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a b

c d Figure-2: (a) Agarose gel of conventional polymerase chain reaction for detection of 16S gene using Mycoplasma genus- specific primer at amplicon size 278 bp. Lane M (Molecular weight marker, 100-1000 bp); Lanes 1-14, positive samples except for lane 3 negative control. (b) Agarose gel of conventional polymerase chain reaction for detection of Mycoplasma arginini species at amplicon size 525 bp. Lane M (Molecular weight marker, 100-1000 bp); Lanes 1-10 positive. (c) Agarose gel of conventional polymerase chain reaction for detection of Mycoplasma ovipneumoniae at amplicon size 418 bp. Lane M, (Molecular weight marker, 100-1000 bp); lanes 1-4 positive samples.

a

b Figure-3: (a) Phylogenetic analysis of Mycoplasma arginini isolates based on 16S gene sequences, MH685445 isolate under study, KP972459 and KP972459 strains isolated from goats in Egypt, HQ661830 and HQ661829 strains from sheep in South Africa, LC158832 from sheep in Japan, and KX2304770 from goat in China. (b) Phylogenetic analysis of M. ovipneumoniae isolates based on 16S gene sequence, KU870650 and KU870647 strains isolated from goats in China, JN257120, EF687778, DQ0000588, and JN257121 from sheep in China, NR_025989 from wild sheep (Ovis aries) in the USA and EU265779 from in the USA and KJ433280 from Norwegian Muskov in Norway. goats including different respiratory manifestations, to isolation, identification, and classification of dif- joint affection, conjunctivitis, and mastitis. The most ferent Mycoplasmas species; Mycoplasma were iso- common species isolated from small ruminants are lated from 24 samples identified biochemically using M. mycoides cluster as Mycoplasma capricolum sub- digitonin, confirmed using PCR as Mycoplasma using species capripneumoniae the causative agent of conta- Mycoplasma genus-specific primers. gious caprine pleuropneumonia, other species isolated The results obtained in this study showed that from sheep and goats including Mycoplasma ovipneu- the prevalence rate of Mycoplasma species was higher moniae [24]; however, M. arginini has been isolated in goats than sheep. In Egypt, similar finding has from a different pathological condition in small rumi- been reported by Ammar et al. [11]; the occurrence nants including pneumonia [17,25]. of Mycoplasma was more frequently in young ani- In this study, 335 samples including lung tis- mals than adult; these findings are closely related to sue, nasal swab, tracheal bifurcation, and bronchial Elshafay et al. [26] who found higher percent of isola- wash were collected from sheep and goats in Egypt tion in young animals than adult, especially in goats in

Veterinary World, EISSN: 2231-0916 668 Available at www.veterinaryworld.org/Vol.12/May-2019/6.pdf Giza and Dakahlia Governorates. Analysis of obtained data on seasonal occurrence of Mycoplasma revealed higher occurrence in winter months (cold weather) than in summer months (hot weather) may be due to 0 0 0

0.41 0.41 1.63 2.44 3.37 1.12 4.45 2.98 increasing stress factor at cold weather and grouping of animals and direct contact with each other. Ten Percentage of

undifferentiated samples were confirmed as M. arginini (2.98%) and Mycoplasma species four samples as M. ovipneumoniae (1.19%) by PCR using specific primer for each species, these results disagreed with Elshafay et al. [26] who recorded higher infection rates. 1 0 1 4 6 3 0 1 0 4 10 The high-frequency rate of isolation of M. arginini

species from different pathological cases in small ruminants, Number of Mycoplasma especially from pneumonic cases, and cases suffer undifferentiated from different respiratory manifestations [7,11,17,25] may refer to its pathogenic role in respiratory affections. Experimental infection of goats <1-year aged ‑ positive by M. arginini isolates formed by Chinedu et al. [6] indicates that M. arginini have some pathogenic effect 0 0 0 0 1.2 0.41 0.41 0.41 1.22 1.12 1.12 as all experimental animals suffered from cough and nasal discharges, on postmortem examination Percentage of

samples by PCR mild-to-severe congestion of lungs with lung edema. Histopathological abnormalities observed were acute

M. ovipneumoniae interstitial pneumonia, hyperemia of pulmonary cap-

illaries with sever infiltration with leucocytes in the interstitial tissues, and alveoli. For estimation, the relationship between M. arginini and M. ovipneumoniae isolated from 1 1 0 1 3 1 0 0 0 1 4 different species and different countries one puri- samples

Number of fied isolate of each species from sheep pneumonic PCR ‑ positive lungs was submitted for partial sequencing of 16S M. ovipneumoniae gene which showed ˃ 99% sequence identity with different M. arginini and M. ovipneumoniae isolates from different species (sheep, goats, and wild rumi- nants) and different countries, phylogenetic analy- ‑ positive

0 0 0 sis of 16S gene showed very high sequence identity 4.5 0.81 0.41 1.22 2.44 3.37 1.19 2.98 between different strains regardless their geographi- cal distribution. Percentage of samples by PCR

M. arginini M. Conclusion

It was concluded that although the respiratory problem in small is very complex, as many Mycoplasma arginine, M. ovipneumoniae=Mycoplasma ovipneumoniae 2 0 1 3 6 3 0 0 1 4 10 = factors play a role including infectious agents and

species using PCR. predisposing factors, the high-frequency isolation of samples Number of M. arginini

PCR ‑ positive M. arginini from different respiratory manifestation and pneumonic lungs suspect its pathogenic role. M. arginine Authors’ Contributions 7 1 2 2 Mycoplasma 58 16 84 89 165 246 335 MMA and FAS designed the experiment proto- samples

Number of col and the study. ESA and SAM collected and ana- lyzed the samples. All authors were involved in data analysis, scientific discussion, and writing of the manuscript. All authors read and approved the final manuscript. Type of sample Nasal swab Bronchial wash Tracheal bifurcation Lung Nasal swab Bronchial wash Tracheal bifurcation Lung Typing of different Typing Acknowledgments The study is funded by the Faculty of Veterinary

Table-4: Animal species Sheep Total Goats Total Total chain reaction, PCR=Polymerase Medicine, Cairo University, Egypt.

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